Various publications, including patents, published applications and scholarly articles, are cited throughout the present specification. The entire contents of each of these publications are incorporated herein, in their entireties. Citations not fully set forth within the specification may be found at the end of the specification.
A key step in coffee processing is the roasting of the green grain. The roasting step is usually carried out in the range of 170° to 230° C. for 5 to 15 minutes and it is responsible for generating most of the aroma, flavor, and color associated with the coffee beverage (Yeretzian, et al., 2005). Depending on the degree of roasting, from 12-40% of the polysaccharides can be degraded at this step (Redgwell, et al., 2002). The roasting step has been reported to alter the length of many of the complex polysaccharide polymers, which can increase their solubility (Redgwell, et al., 2002). Fragmentation of the coffee polysaccarides is thought to favourably affect beverage organoleptic properties such as mouthfeel (Illy and Viani 1995) and foam stability (Nunes, et al., 1997). Breakdown of the polysaccharides is also thought to influence the binding of volatile aroma compounds indirectly because some complex carbohydrate degradation products participate in the formation of the roasted grain melanoidins, a class of poorly defined compounds that constitute over 20% of the roasted grain dry weight (Charles-Bernard, et al., 2005). The roasting induced cleavage of the polysaccharides may also produce an increase in the amount of solids extracted from the coffee grain, a property of critical importance for the production of soluble coffee. Additionally, the fragmentation/degradation of the carbohydrates in the coffee grain also contribute to the generation of an important group of coffee flavor and aroma molecules via the Maillard reaction associated with coffee roasting (Yeretzian, et al, 2005).
Carbohydrates make up a large proportion of the mature green coffee grain (green bean). Approximately 48-55% of the dry weight in arabica (Coffea arabica) and robusta (C. canephora) green grain is composed of carbohydrate, some of which is in the form of complex polysacchaccarides, while other forms include free mono- and di-saccharides (Clifford M. N., 1985 In Coffee: Botany, Biochemistry, and Production, pp 374, ed. Clifford, M. and Willson, K., Croom Melm Ltd, London; Fischer, et al. 2001, Carbohydrate Research, 330, 93-101). Three main types of complex carbohydrate-based polymers have been identified in the coffee grain. The most abundant grain polysaccharides are the galactomannans, which are reported to represent up to 25% of the mass in the mature green coffee grain, i.e., approximately 50% of the grain carbohydrates. (Oosterveld et al., 2003 Carbohydrate Polymers 52, 285-2960). The next most abundant group of polysaccharides are the arabinogalactans which comprise up to 35% of the green grain polysaccharides (Oosterveld et al., 2003, supra). The remaining approximately 16% of the Arabica green grain polysaccharides consist primarily of cellulose and xyloglucans (Oosterveld et al., 2003)
Mannan containing hemicelluloses are composed of a backbone of beta 1-4 linked mannose molecules, and although they can be widely found in plants the mannans have been considered to be a relatively minor constituent in the walls of most plant cell types (Bacic, Harris, and Stone 1988; Fry 2004; Somerville, et al., 2004b). Some endosperm containing seeds, such as those of Leguminosae, Palmae, and the commercially important Coffea species, have quite large amounts of galactomannans in the seed endosperm cell walls (Matheson 1990; Buckeridge, et. al., 2000; Pettolino, et al., 2001; Redgwell, et al., 2002; Hanford, et al., 2003). Galactomannans are characterized by mannan chains that have single galactosyl molecules attached by a (1-6) alpha linkage. The galactomannans of the seed endosperm appear to be associated with the secondary cell wall thickening of the endosperm cell wall (Pettolino, et al., 2001; Sunderland, et al., 2004; Somerville, et al., 2004a) and are believed to form part of the energy reserve of the mature seed, which is analogous to role played by starch in cereal endosperms (Reid 1985). Other functions that have been theorized for the endosperm galactomannans include facilitating imbibition/germination and the protection of the seed embryo from dessication (Reid and Bewley 1979). Other main mannan based cell wall polymers include the glucomannans which have some of the mannose units substituted by beta-1,4-linked glucose residues, and the galactoglucomannans which are glucomannans with alpha-1,6-linked galactose residues. Galactoglucomannans with low levels of galactose are important constituents of thickened lignified secondary cell walls of gymnosperms (Lundqvist, J., et al., 2002) and have also been found in kiwi fruit (Actinidia deliciosa) and tissue cultured tobacco (Nicotiana plumbaginifolia) cells (Schroder, R., et al., 2001; Sims, I., et al., 1997). Recently studies have purported that mannan polymers exist in the thickened secondary cell walls of xylem elements, xylem parenchyma and interfasicular fibers of the model angiosperm Arabidopsis thaliana (Handford et al 2003). They also detected significant levels of mannans in the thickened epidermal cell walls of leaves and stem, and lower levels of mannans in most other tissues examined indicating the widespread presence of mannans in arabidopsis.
While the cellulose polymers are known to be synthesized at the plasma membrane, most non-cellulosic polysaccharides are believed to be made in the golgi apparatus and then transported outside the cell membrane into the apoplastic space (Keegstra and Raikhel 2001; Somerville, Bauer, Brininstool, Facette, Hamann, Milne, Osborne, Paredez, Persson, Raab, Vorwerk, and Youngs 2005; Liepman, Wilkerson, and Keegstra 2005b). Two membrane bound glycosyltransferases are known to be involved in synthesizing the galactomannans: a Mg++0 dependant, GDP-Man dependant (1,4)-beta-D-mannosyltransferase or mannan synthase (MS) and a Mn++ dependant, UDP-Gal dependant mannan specific (1,6)-alpha-D-galactosyltransferase (GMGT), and these enzymes are believed to work together very closely to determine the statistical distribution of galactosyl residues along the mannan polymer (Edwards, Choo, Dickson, Scott, Gridley, and Reid 2004). Confirmation that mannans are synthesized in the golgi apparatus has recently been obtained by using mannan specific antibodies to detect mannan synthesis in vitro, and this further supports the overall model in which the hemicellulose type polysaccharides such as the galactomannans are made in the golgi and then transported to the cell membrane and secreted into the apoplast region (Handford, Baldwin, Goubet, Prime, Miles, Yu, and Dupree 2003; Somerville, Bauer, Brininstool, Facette, Hamann, Milne, Osborne, Paredez, Persson, Raab, Vorwerk, and Youngs 2005). The importance of a golgi bound GMGT protein in the synthesis of seed endosperm galactomannans, and more precisely in controlling the level of galactose modification, has recently been demonstrated by showing that either over-, or under-expressing the Lotus japonicus GMGT protein causes predicable changes in the galactose/mannose ratios in the seed (Edwards, Choo, Dickson, Scott, Gridley, and Reid 2004).
Until recently, the genes responsible for the synthesis of the plant cell mannans were not known. The first gene isolated that encodes a biochemically demonstrated mannan synthase was the ManS from Cyamopsis tetragonoloba (guar) seeds (Dhugga, et al., 2004). The cDNA for CtManS was isolated from EST libraries made from three different seed developmental stages of guar, a seed which makes very large quantities of galactomannans. The CtManS related ESTs were identified by searching for sequences with strong similarities to plant CelA (cellulose synthases generating beta-1,3-glucans) and Csl (cellulose synthase-like proteins). The Csl genes have significant similarity to the CelA genes, and have been previously proposed as candidate genes for enzymes involved in the synthesis of hemicelluoses like galactomannans (Cutler and Somerville 1997; Richmond and Somerville 2000; Hazen, et al., 2002). The abundance of the candidate mannan synthase ESTs in each guar seed library corresponded to the levels of mannan synthase activity biochemically measured at each stage, suggesting these ESTs represented a mannan synthase. The putative guar mannan synthase cDNA was shown to encode a functional enzyme by showing that soybean somatic embryos, which normally have no detectable mannan synthase activity, exhibited significant mannan synthase activity when they over-express the CtManS cDNA sequence (Dhugga, et al., 2004). The functional recombinant enzyme was found to be located in the golgi apparatus. In the arabidopsis genome, there are over 25 genes annoted as Csl genes and these are subdivided into families based on their sequence homologies. Recently, a functional evaluation has been carried out on recombinant proteins generated from a number of the arabidopsis Csl gene sequences and it was determined that several members of the CslA gene family encoded proteins with beta-mannan synthase activity (Liepman, et al., 2005).
There is little information available directed to the metabolism of mannan related polymers in coffee. Several highly related cDNA encoding an alpha-galalactosidase found in coffee grain have been obtained and the expression of this gene in developing grain indicates that this gene is induced during the formation and expansion of the endosperm (approximately 22-27 WAF (Weeks After Fertilization) and expression can also be detected in leaves, flowers, zygotic embryos, and weakly in roots (Marraccini, et al., 2005). The galactose/mannose ratio of the coffee grain galactomannans falls from a ratio of approximately 1:2 to 1:7 at an early stage of grain development (11 WAF; weeks after fertilization) to a ratio of 1:7 to 1:40 near maturity at 31 WAF (Redgwell, et al., 2003). This information, together with the developmental expression data for the alpha-galactosidase presented above, led to the theory that this particular alph-galactosidase gene product could be directly involved in lowering the galactose content of the coffee grain galactomannans that begin around 21-26 WAF and continues to grain maturity (Redgwell, et al., 2003). Support for this model was found in the developing seeds of senna (Senna occidentalis) where a significant increase in alpha-galactosidase activity was found to coincide with the reduction of the galactose content of seed galactomannans (Edwards, et al., 1992). Further support for the involvement of an alpha-galactosidase in the reduction of the galactose content was subsequently obtained when the senna alpha-galactosidase was expressed in developing Cyamopsis tetragonoloba (guar) seeds with the aid of a seed specific promoter (Joersbo, et al., 2001). Guar seeds normally have high levels of galactomannans that possess a very high galactose/mannan ratio, but guar seeds produced from the plants expressing senna alpha-galactosidase showed significant reductions in the level of galactose/mannose ratio in the modified guar seeds. Two cDNA encoding distinct endo-beta mannanases (manA and manB) have also been isolated from germinating coffee grain (Marraccini, et al., 2001). The corresponding genes were not expressed in the developing grain, but both were expressed during germination, with transcripts being detected starting at 10-15 days after imbibition. This observation suggests that both of these mananases are associated with the degradation of galactomannans during germination and result in the liberation of free sugars that then act as both a source of energy and reduced carbon for the germinating seed. The expression of manA was examined and no expression was detected in leaves, somatic embryos, flower buds or roots (Marraccini, et al., 2001).
Despite the abundance of galactomannans in coffee grain and the implicit importance of enzymes that participate in galactomannan synthesis, little information is available on these genes in coffee. Thus, there is a need to identify, isolate and characterize the enzymes, genes, and genetic regulatory elements involved in the galactomannan biosynthetic pathway in coffee. Such information will enable galactomannan synthesis to be genetically manipulated, with the goal of imparting desirable phenotypic advantages associated with altered galactomannan production.